Paul I. Freimuth

Research Interests

Our current studies focus on toxic gain-of-function by proteins that misfold
during recombinant expression in microbial cells. Errors in folding of
endogenous proteins occur frequently, and cells have several independent
quality control pathways to monitor protein folding status and mitigate the
potentially toxic effects associated with accumulation of misfolded protein
species. Failure of these QC pathways to restore protein folding homeostasis
can result in cell death and disease. A number of factors in addition to
amino acid sequence can influence folding, including temperature
fluctuations, global and local rates of translation elongation, and
availability of molecular chaperones and other enzymes for
post-translational modification. These factors may be changed radically when
polypeptides are expressed in heterologous host cells using recombinant
expression technology, which likely contributes to the frequent production
of misfolded protein species in such experiments. Misfolded polypeptides
produced by these methods often accumulate as insoluble aggregates that have
little or no apparent deleterious effects on cell physiology. However, we
have encountered several instances where misfolded proteins gain toxic
functions that strongly inhibit cell growth. In earlier work, for example,
we found that the head domain of the serotype 2 adenovirus fiber protein is
conditionally toxic when expressed in thi- strains of E. coli, and that the
essential vitamin thiamine diphosphate is tightly sequestered in the protein
subunit interface. By contrast, fiber head domains derived from other
adenoviruses closely related to serotype 2 do not bind thiamine diphosphate
and are not toxic for thi- strains. Results of this study provide the
important insight that the toxic activity of misfolded proteins or
misassembled protein oligomers can result from highly specific mechanisms.
The toxicity of amyloid fibrils, in contrast, has been proposed to result
from nonspecific sequestration of numerous essential cell proteins.

The conclusion that the toxic activity of misfolded proteins can result from
highly specific mechanisms is also supported by our current investigation of
the toxic activity of the Arabidopsis Gld protein, which misfolds during
expression in E. coli. Like the fiber head domain, the toxic activity of Gld
is conditional, in this case depending on which RNA polymerase is used for
expression of the plasmid-borne Gld gene in E. coli. When gld is transcribed
by the E. coli RNA polymerase, cell growth is arrested immediatedly
following induction of expression, and cell viability plummets. Minute but
detectable quantities of Gld protein are produced in growth-arrested cells.
Importantly, the Gld protein is found in the soluble fraction of cell
lysates. When gld is transcribed by the T7 phage RNA polymerase, by
contrast, cell growth proceeds following induction, and Gld protein
accumulates to high concentration in cells as insoluble aggregates. These
results are in striking parallel to recent studies of amyloidogenic
proteins, which show that toxicity is associated primarily with soluble,
pre-aggregated oligomers of misfolded protein, whereas the insoluble amyloid
fibrils themselves were found to be relatively nontoxic. Additional studies
indicate that an internal region 26-residues in length is both necessary and
sufficient for the toxic activity of the 325-residue Gld polypeptide. The
corresponding synthetic peptide inhibits transcription in vitro by purified
E. coli RNA polymerase. Results of our current study thus support the
conclusion that the toxic activity of misfolded Gld also results from a
highly specific mechanism, in this case inhibition of transcription by E.
coli RNA polymerase. Our results also provide the insight that the
aggregation-prone Gld polypeptide can only exert its toxic effect over short
distances, for example only when the host RNA polymerase is physically
coupled to ribosomes translating the Gld polypeptide. In future studies we
will determine the molecular mechanism of the toxic Gld peptide interaction
with RNA polymerase.

One of ten national laboratories overseen and primarily funded by the Office of Science of the
U.S. Department of Energy (DOE), Brookhaven National Laboratory conducts research in the physical,
biomedical, and environmental sciences, as well as in energy technologies and national security.
Brookhaven Lab also builds and operates major scientific facilities available to university, industry
and government researchers. Brookhaven is operated and managed for DOE's Office of Science by Brookhaven
Science Associates, a limited-liability company founded by the Research Foundation for the State
University of New York on behalf of Stony Brook University, the largest academic user of Laboratory
facilities, and Battelle, a nonprofit applied science and technology organization.